Imaging mitochondria through bone in live mice using two-photon fluorescence microscopy with adaptive optics
IntroductionMitochondria are extremely important organelles in the regulation of bone marrow and brain activity. However, live imaging of these subcellular features with high resolution in scattering tissues like brain or bone has proven challenging.MethodsIn this study, we developed a two-photon fl...
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Frontiers Media S.A.
2023-02-01
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| Series: | Frontiers in Neuroimaging |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fnimg.2023.959601/full |
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| author | Tianyi Zheng Adrian R. Liversage Kayvan F. Tehrani Jarrod A. Call Peter A. Kner Luke J. Mortensen Luke J. Mortensen |
| author_facet | Tianyi Zheng Adrian R. Liversage Kayvan F. Tehrani Jarrod A. Call Peter A. Kner Luke J. Mortensen Luke J. Mortensen |
| author_sort | Tianyi Zheng |
| collection | DOAJ |
| description | IntroductionMitochondria are extremely important organelles in the regulation of bone marrow and brain activity. However, live imaging of these subcellular features with high resolution in scattering tissues like brain or bone has proven challenging.MethodsIn this study, we developed a two-photon fluorescence microscope with adaptive optics (TPFM-AO) for high-resolution imaging, which uses a home-built Shack-Hartmann wavefront sensor (SHWFS) to correct system aberrations and a sensorless approach for correcting low order tissue aberrations.ResultsUsing AO increases the fluorescence intensity of the point spread function (PSF) and achieves fast imaging of subcellular organelles with 400 nm resolution through 85 μm of highly scattering tissue. We achieved ~1.55×, ~3.58×, and ~1.77× intensity increases using AO, and a reduction of the PSF width by ~0.83×, ~0.74×, and ~0.9× at the depths of 0, 50 μm and 85 μm in living mouse bone marrow respectively, allowing us to characterize mitochondrial health and the survival of functioning cells with a field of view of 67.5× 67.5 μm. We also investigate the role of initial signal and background levels in sample correction quality by varying the laser power and camera exposure time and develop an intensity-based criteria for sample correction.DiscussionThis study demonstrates a promising tool for imaging of mitochondria and other organelles in optically distorting biological environments, which could facilitate the study of a variety of diseases connected to mitochondrial morphology and activity in a range of biological tissues. |
| first_indexed | 2024-04-10T09:57:49Z |
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| id | doaj.art-950a1e742f774c0f8965dfd0135aa849 |
| institution | Directory Open Access Journal |
| issn | 2813-1193 |
| language | English |
| last_indexed | 2024-04-10T09:57:49Z |
| publishDate | 2023-02-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Neuroimaging |
| spelling | doaj.art-950a1e742f774c0f8965dfd0135aa8492023-02-16T11:59:13ZengFrontiers Media S.A.Frontiers in Neuroimaging2813-11932023-02-01210.3389/fnimg.2023.959601959601Imaging mitochondria through bone in live mice using two-photon fluorescence microscopy with adaptive opticsTianyi Zheng0Adrian R. Liversage1Kayvan F. Tehrani2Jarrod A. Call3Peter A. Kner4Luke J. Mortensen5Luke J. Mortensen6School of Electrical and Computer Engineering, University of Georgia, Athens, GA, United StatesSchool of Chemical, Materials and Biomedical Engineering, University of Georgia, Athens, GA, United StatesBiophotonics Imaging Laboratory, The University of Illinois Urbana-Champaign, Urbana, IL, United StatesDepartment of Physiology and Pharmacology, University of Georgia, Athens, GA, United StatesSchool of Electrical and Computer Engineering, University of Georgia, Athens, GA, United StatesSchool of Chemical, Materials and Biomedical Engineering, University of Georgia, Athens, GA, United StatesRegenerative Bioscience Center, Rhodes Center for ADS, University of Georgia, Athens, GA, United StatesIntroductionMitochondria are extremely important organelles in the regulation of bone marrow and brain activity. However, live imaging of these subcellular features with high resolution in scattering tissues like brain or bone has proven challenging.MethodsIn this study, we developed a two-photon fluorescence microscope with adaptive optics (TPFM-AO) for high-resolution imaging, which uses a home-built Shack-Hartmann wavefront sensor (SHWFS) to correct system aberrations and a sensorless approach for correcting low order tissue aberrations.ResultsUsing AO increases the fluorescence intensity of the point spread function (PSF) and achieves fast imaging of subcellular organelles with 400 nm resolution through 85 μm of highly scattering tissue. We achieved ~1.55×, ~3.58×, and ~1.77× intensity increases using AO, and a reduction of the PSF width by ~0.83×, ~0.74×, and ~0.9× at the depths of 0, 50 μm and 85 μm in living mouse bone marrow respectively, allowing us to characterize mitochondrial health and the survival of functioning cells with a field of view of 67.5× 67.5 μm. We also investigate the role of initial signal and background levels in sample correction quality by varying the laser power and camera exposure time and develop an intensity-based criteria for sample correction.DiscussionThis study demonstrates a promising tool for imaging of mitochondria and other organelles in optically distorting biological environments, which could facilitate the study of a variety of diseases connected to mitochondrial morphology and activity in a range of biological tissues.https://www.frontiersin.org/articles/10.3389/fnimg.2023.959601/fulltwo-photon fluorescence microscopyaberrationadaptive opticsmitochondriamouse cranial bone |
| spellingShingle | Tianyi Zheng Adrian R. Liversage Kayvan F. Tehrani Jarrod A. Call Peter A. Kner Luke J. Mortensen Luke J. Mortensen Imaging mitochondria through bone in live mice using two-photon fluorescence microscopy with adaptive optics Frontiers in Neuroimaging two-photon fluorescence microscopy aberration adaptive optics mitochondria mouse cranial bone |
| title | Imaging mitochondria through bone in live mice using two-photon fluorescence microscopy with adaptive optics |
| title_full | Imaging mitochondria through bone in live mice using two-photon fluorescence microscopy with adaptive optics |
| title_fullStr | Imaging mitochondria through bone in live mice using two-photon fluorescence microscopy with adaptive optics |
| title_full_unstemmed | Imaging mitochondria through bone in live mice using two-photon fluorescence microscopy with adaptive optics |
| title_short | Imaging mitochondria through bone in live mice using two-photon fluorescence microscopy with adaptive optics |
| title_sort | imaging mitochondria through bone in live mice using two photon fluorescence microscopy with adaptive optics |
| topic | two-photon fluorescence microscopy aberration adaptive optics mitochondria mouse cranial bone |
| url | https://www.frontiersin.org/articles/10.3389/fnimg.2023.959601/full |
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